The lack of adequate animal models of Cystic Fibrosis (CF) has hindered progress toward understanding CF lung pathophysiology and the development and testing of gene-based and pharmacologic therapies. Although the field has learned a great deal from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mouse models, this species has been unable to model the natural progression of human CF lung disease. In part, this limitation appears to stem from a large divergence in airway cell biology and varying composition of alternative chloride channels in the airway epithelium between mice and humans. The generation of alternative animal models of CF that mimic the human condition is paramount to both basic and clinical CF research. Without such a model, pathophysiologic hypotheses are extremely difficult for translate from in vitro basic molecular mechanisms to in vivo multicellular processes which determine the progression of lung disease in CF. Furthermore, without an authentic animal model of CF lung disease, the time frame required for testing clinical therapies capable of preventing the progression of CF lung disease is considerably longer. This proposal will attempt to fill this gap in available animal models of CF lung disease by generating a CF ferret. The ferret is an ideal choice for such a CF model since its lung physiology and airway cell biology closely resembles that of humans. For example, ferrets and humans share all the major cell types in the airway including basal, ciliated, goblet, and intermediate cells as well as having similar distributions of submucosal glands. Over the past three years, our laboratory has made considerable progress in moving toward the goal of generating a CF ferret model. Our approach to generating this model utilizes recently developed somatic-cell embryo cloning techniques together with allele-specific gene targeting in somatic cells. Both embryonic stem cells and fetal fibroblasts will be evaluated as competent nuclear donors for cloning ferrets and several strategies for targeting the ferret CFTR will be tested in these cell types. Gene targeting approaches will include methods of mismatch DNA repair to introduce the G551D mutation into the CFTR gene and homologous recombination to create null mutation deleting a portion of exon 10 of CFTR. In addition to completing the necessary steps for generating a CF ferret model, this proposal will also seek to understand the importance of submucosal glands in preventing airway infection and bacterial biofilm formation in the ferret airway. Submucosal glands in the cartilaginous airways of humans and ferrets express very high levels of CFTR mRNA and protein. This similarity is an important conceptual feature for choosing this species as a model for CF. Using ferret tracheal xenografts with and without submucosal glands, we will seek to better understand the importance of CFTR in regulating glandular secretion of antibacterial factors and preventing airway bacterial colonization. Such studies will begin to provide a working foundation for utilizing the CF ferret model once it is available.